Derivados porfirínicos e splicing alternativo do mRNA induzido por terapia fotodinâmica

Mestrado em QuímicaNeste trabalho é descrita a síntese de porfirinas regulares e N-confusas com o objetivo da realização de estudos acerca da sua eficiência como fotossensibilizadores (PSs) em terapia fotodinâmica (PDT) e a influência desta no splicing alternativo do mRNA. Os derivados porfirinicos foram preparados tendo como estrutura base a 5,10,15,20-tetraquis(1-metilpiridinio-4-il)porfirina. Inicialmente irá ser realizada uma breve introdução sobre os macrociclos tetrapirrólicos, nomeadamente sobre porfirinas. Segue-se o capítulo 2 onde há a descrição sintética dos derivados porfirínicos usados neste trabalho. Desta forma, através das condições estabelecidas por Lindsey foram sintetizadas duas porfirinas: 5,10,15,20-tetraquis(4-bromometilfenil)-2-aza-21-carboporfirina e a 5,10,15,20-tetraquis(4-bromometilfenil)porfirina e procedendo-se à sua cationização com piridina que originou os derivados 5,10,15,20-tetraquis(4-(piridinio-1-il-metil)fenil)-2-aza-21-carboporfirina e 5,10,15,20-tetraquis(4-(piridinio-1-il-metil)fenil)porfirina. A formação das porfirinas neutras ocorreu com baixo rendimento, mas a sua cationização foi conseguida com rendimentos acima dos 80%. Neste mesmo capítulo ainda são apresentadas as caracterizações fotofísicas de todos os derivados porfirínicos, nomeadamente fluorescência e geração de oxigénio singleto. As porfirinas regulares foram aquelas que demonstraram bons rendimentos quânticos de fluorescência e capacidade para gerar oxigénio singleto em DMF. No capítulo 3 são descritos todos os estudos biológicos realizados neste trabalho. Inicialmente foram realizados estudos de viabilidade celular em células MCF-7 e HeLa usando o teste com WST-1 e ATPliteTM e como PS de referência a 5,10,15,20-tetraquis(1-metilpiridinio-4-il)porfirina. Foram realizados estudos de concentração (0.1 μM-5.0 μM) e tempo de irradiação (0, 5, 10 e 15 min). A 5,10,15,20-tetraquis(4-(piridinio-1-il-metil)fenil)porfirina revelou ser o melhor PS, tendo sido selecionado um tempo de irradiação de 15 min e uma concentração até 5 μM para o estudos subsequentes. Os estudos de localização subcelular e uptake revelaram que a 5,10,15,20-tetraquis(4-(piridinio-1-il-metil)fenil)porfirina entra nas células mas não se localiza no núcleo como acontece com o controlo positivo. São ainda descritos estudos acerca do tipo de morte celular que as células podem sofrer depois da PDT com estes PSs. Por fim os estudos da influência da terapia fotodinâmica no splicing alternativo mostrou que a 5,10,15,20-tetraquis(4-(piridinio-1-il-metil)fenil)porfirina e a 5,10,15,20-tetraquis(1-metilpiridinio-4-il)porfirina parecem induzir um ligeiro splicing alternativo do mRNA.In this work the synthesis of regular and N- confused porphyrins with the objective of assessing its efficiency as photosensitizers (PSs) in photodynamic therapy (PDT) and its influence in alternative splicing of mRNA is described. The porphyrin derivatives were prepared having as a template the 5,10,15,20-tetrakis(1-methylpyridynium-4-yl)porphyrin. Initially, a brief introduction about tetrapyrrolic macrocycles, namely about porphyrins will be done. Then, chapter 2 will follow with the synthetic description of the porphyrin derivatives used in this work. Through Lindsey conditions, two porphyrins were synthesized: 5,10,15,20-tetrakis(4-bromomethylphenyl)-2-aza-21-carbaporphyrin; 5,10,15,20-tetrakis(4-bromomethylphenyl)porphyrin and then we proceeded to their cationization with pyridine to give: 5,10,15,20-tetrakis(4-(pyridynium-1-yl-methyl)phenyl)-2-aza-21-carbaporphyrin and 5,10,15,20-tetrakis(4-(pyridynium-1-yl-methyl)phenyl)porphyrin. The formation of the neutral porphyrins occurred with low yield but its cationization was achieved with yields higher than 80%. In the same chapter, the photophysical characterization of all porphyrins, namely singlet oxygen production and fluorescence quantum yield were described. The regular porphyrins demonstrated good fluorescence quantum yields and ability to generate singlet oxygen in DMF. In chapter 3, all the biological assays done in this work are described. Initially, cellular viability studies were done in MCF-7 and HeLa cells using the WST-1 reagent and ATPliteTM and as PSs reference the 5,10,15,20-tetrakis(1-methylpyridynium-4-yl)porphyrin. Studies of concentration (0.1 μM-5.0 μM) and irradiation time (0, 5, 10 and 15 min) were also done. The 5,10,15,20-tetrakis(4-(pyridynium-1-yl-methyl)phenyl)porphyrin showed to be the best PS and 15 min irradiation and a concentration up to 5 μM for the following studies were selected. The subcellular localization and uptake studies revealed that 5,10,15,20-tetrakis(4-(pyridynium-1-yl-methyl)phenyl)porphyrin can enter the cells but it is not localized in the nucleus, contrary to 5,10,15,20-tetrakis(1-methylpyridynium-4-yl)porphyrin. The studies regarding type of cell death that cells undergo after PDT treatment were also assessed. At last, the 5,10,15,20-tetrakis(4-(pyridynium-1-yl-methyl)phenyl)porphyrin and 5,10,15,20-tetrakis(1-methylpyridynium-4-yl)porphyrin seemed to induce a small alternative splicing of mRNA

Stacked Denoising Autoencoders and Transfer Learning for Immunogold Particles Detection and Recognition

In this paper we present a system for the detection of immunogold particles and a Transfer Learning (TL) framework for the recognition of these immunogold particles. Immunogold particles are part of a high-magnification method for the selective localization of biological molecules at the subcellular level only visible through Electron Microscopy. The number of immunogold particles in the cell walls allows the assessment of the differences in their compositions providing a tool to analise the quality of different plants. For its quantization one requires a laborious manual labeling (or annotation) of images containing hundreds of particles. The system that is proposed in this paper can leverage significantly the burden of this manual task. For particle detection we use a LoG filter coupled with a SDA. In order to improve the recognition, we also study the applicability of TL settings for immunogold recognition. TL reuses the learning model of a source problem on other datasets (target problems) containing particles of different sizes. The proposed system was developed to solve a particular problem on maize cells, namely to determine the composition of cell wall ingrowths in endosperm transfer cells. This novel dataset as well as the code for reproducing our experiments is made publicly available. We determined that the LoG detector alone attained more than 84\% of accuracy with the F-measure. Developing immunogold recognition with TL also provided superior performance when compared with the baseline models augmenting the accuracy rates by 10\%

An insight on the role of photosensitizer nanocarriers for Photodynamic Therapy

Photodynamic therapy (PDT) is a modality of cancer treatment in which tumor cells are destroyed by reactive oxygen species (ROS) produced by photosensitizers following its activation with visible or near infrared light. The PDT success is dependent on different factors namely on the efficiency of the photosensitizer deliver and targeting ability. In this review a special attention will be given to the role of some drug delivery systems to improve the efficiency of tetrapyrrolic photosensitizers to this type of treatment.publishe

Photodynamic inactivation of a RNA-virus model using water-soluble β-octa-Substituted pyridinium-pyrazolyl phthalocyanines

Among the various groups of microorganisms, viruses have generally a greater capacity for mutation, especially RNA viruses, as was demonstrated by SARS-CoV2 virus mutations. This high mutation rate promotes the development of their resistance to traditional antivirals and establishes the resistance behaviour in virus populations, decreasing their susceptibility to these drugs. In this context, the photodynamic treatment appears as a potentially effective method against microorganisms and, considering its mode of action is not likely to lead to the development of resistance. In this work, two newly zinc(II) phthalocyanines (ZnPcs) bearing pyridinium-pyrazolyl groups (2a and 3a) were synthesized, characterized, and applied in photodynamic inactivation (PDI) of bacteriophage Φ6 (or Phage Phi6) as a RNA-virus model. These quaternized dyes were applied at different concentrations (from 5.0 to 20 μM, and under white light irradiation in the irradiance range between 50 and 150 mW/cm2) to test their efficiency for possible clinical or environmental applications. The results showed that the new cationic ZnPcs 2a and 3a efficiently inactivate the RNA-virus model (bacteriophage Φ6), even at the lowest tested irradiance. These compounds are thus promising photosensitizers to be used in various contexts.info:eu-repo/semantics/publishedVersio

Dérivés des Porphyrines Tétracationique dans le cancer du sein

Photodynamic therapy (PDT) is an alternative to conventional chemotherapy for the treatment of several types of cancer. Its advantages are reduced side effects and development of resistance mechanisms. In this work, we evaluated the photosensitization capabilities of 5,10,15,20-tetrakis[4-(pyridinium-1-yl-methyl)phenyl]porphyrin, its N-confused isomer and tow if its neutral precursors. The results were compared with the ones obtained with the cationic 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP) used as positive control. Both regular porphyrin derivatives showed higher efficiency to generate singlet oxygen than TMPyP. Next, we demonstrated that one of the cationic porphyrin is an efficient photosentitizer kills MCF7 breast cancer cells. The study of the cell death mechanisms induced by the photodynamic process showed that 5,10,15,20-tetrakis[4-(pyridinium-1-yl-methyl)phenyl]porphyrin and TMPyP caused cell death by autophagic flux and necrosis

Tetracationic porphyrin derivatives against human breast cancer

Photodynamic therapy (PDT) is an approved therapeutic approach and an alternative to conventional chemotherapy for the treatment of several types of cancer with the advantages of reducing the side effects and developing resistance mechanisms. Here, was evaluated the photosensitization capabilities of 5,10,15,20-tetrakis[4-(pyridinium-1-yl-methyl)phenyl]porphyrin (3), its N-confused isomer (4) and of the neutral precursors (1) and (2) and the results were compared with the ones obtained with the cationic 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP). Both regular porphyrin derivatives 1 and 3 showed higher efficiency to generate singlet oxygen than TMPyP. The PDT assays towards MCF-7 cells under red light irradiation (λ > 640 nm, 23.7 mW cm-2) demonstrated that the cationic porphyrin 3 is an efficient photosensitizer to kill MCF-7 breast cancer cells. The study of the cell death mechanisms induced by the photodynamic process showed that the studied porphyrin 3 and TMPyP caused cell death by autophagic flux and necrosis.publishe

N-confused porphyrin immobilized on solid supports: Synthesis and metal ions sensing efficacy

Thanks are due to FCT/MEC for the financial support to the QOPNA research Unit (FCT UID/QUI/00062/2013), the CESAM (UID/AMB/50017/2013) and CICECO (POCI-01-0145-FEDER-007679 and UID/CTM/50011/2013), through national funds and when applicable co-financed by the FEDER, within the PT2020 Partnership Agreement and "Compete" 2020, and also to the Portuguese NMR Network. Scientific PROTEOMASS Association (Portugal) and the Associate Laboratory for Green Chemistry LAQV (FCT/MEC (UID/QUI/50006/2013) and PT2020-POCI-01-0145-FEDER-007265) for general funding. NMM Moura and ATPC Gomes thank FCT for their postdoctoral grants (SFRH/BPD/84216/2012 and SFRH/BPD/79521/2011, respectively). Tiago Fernandes thanks FCT for the PhD grant (SFRH/BD/130934/2017). Ana L. Daniel-da-Silva acknowledges FCT for the research contract under the Program 'Investigador FCT' 2014.In this work, the N-confused porphyrin 5,10,15,20-tetraphenyl-2-aza-21-carbaporphyrin (NCTPP) was immobilized on neutral or cationic supports based on silica and on Merrifield resin. The new materials were characterized by appropriate techniques (UV-Vis spectroscopy, SEM, and zeta potential analysis). Piezoelectric quartz crystal gold electrodes were coated with the different hybrids and their ability to interact with heavy metals was evaluated. The preliminary results obtained showed that the new materials can be explored for metal cations detection and the modification of the material surface is a key factor in tuning the metal selectivity.publishersversionpublishe